Inteins are unique protein insertion sequences capable of self-excision, enabling the covalent ligation of flanking extein peptides via amide bond formation. This process proceeds spontaneously without requiring external enzymes, cofactors, or chemical reagents, granting inteins exceptional biocompatibility and traceless performance in protein engineering applications. Split inteins represent a specialized and versatile subclass whose splicing domains are encoded by two separate gene fragments rather than a single continuous open reading frame. These fragments, known as the N-terminal (IntN) and C-terminal (IntC) split inteins, associate through non-covalent interactions including hydrophobic forces, hydrogen bonds, and van der Waals forces to assemble into an active three-dimensional structure, which then drives efficient extein ligation and enables protein trans-splicing. Protein trans-splicing mediated by split inteins has become a cornerstone for traceless protein ligation owing to its high specificity and irreversibility, fundamentally reshaping strategies for protein modification, assembly, and functional regulation. Compared with traditional chemical ligation methods, split intein systems require no complex chemical derivatization of peptide fragments and can operate efficiently at micromolar concentrations under physiological conditions, thus avoiding structural and functional damage caused by organic reagents. In contrast to enzymatic ligation tools such as sortase, split inteins eliminate the need for additional enzymes or cofactors, simplifying reaction systems, reducing costs, and minimizing non-specific side products. These distinctive advantages render split inteins highly promising for applications in chemical biology, synthetic biology, and biopharmaceutical development. In recent years, deepened mechanistic understanding has established structure-guided rational design as the primary approach to overcoming key limitations of split inteins, including intrinsic aggregation propensity, strict extein sequence dependence, and limited splicing efficiency. Bioinformatic tools have been used to identify aggregation-prone regions in the IntN fragment, and site-directed mutagenesis of hydrophobic residues, relocation of split sites, or removal of misfolding-prone sequences has substantially reduced in vitro aggregation and improved soluble expression and assembly activity. Rational engineering of catalytic residues and adjacent flexible loops has relaxed strict amino acid preferences at extein junctions, enhancing sequence tolerance and reducing the risk of functional impairment in target proteins. Consensus design based on multiple sequence alignments has yielded ultra-fast splicing variants such as Cfa DnaE and Cat-TerL, which exhibit significantly accelerated kinetics and improved tolerance to denaturing conditions. Meanwhile, advances in structural biology have further clarified the conformational dynamics and catalytic mechanisms of splicing, supporting the precise design of high-performance intein modules. On this basis, electrostatic interaction tuning and metagenomic screening have yielded multiple mutually orthogonal split intein pairs, enabling selective multi-fragment protein ligation and providing new routes for the efficient synthesis of large multi-domain functional proteins. With these engineered split inteins offering continuously improved performance and expanded applicability, protein trans-splicing has been widely applied in numerous cutting-edge areas of protein research and biomedicine. In gene delivery, split intein-based systems overcome the packaging limit of adeno-associated viral vectors, enabling the accurate reconstitution of large therapeutic proteins and base editors in target cells, thereby enhancing the efficacy and scope of gene therapy for genetic diseases. In internal protein sequence editing, split inteins mediate precise sequence replacement and modification in flexible regions or loops of target proteins, without the need for complex multi-step ligation and protein refolding involved in traditional protein semisynthesis. In protein-protein interaction studies, intein-mediated splicing covalently captures transient and weak intracellular complexes, enabling sensitive, high-throughput interaction detection and drug screening. In synthetic biology, conditionally controllable splicing systems support the construction of diverse intracellular and cell-surface biological logic gates for the precise regulation of cellular behavior. In mechanistic biochemical research, split inteins enable photocatalytic proximity labeling and site-specific tagging, allowing the preparation of homogeneous protein samples carrying precise post-translational modifications such as ubiquitination and polyglutamylation for chromatin interactome analysis and epigenetic studies. Moreover, covalent trapping strategies using split inteins stabilize transient enzymatic intermediates, providing unprecedented insights into molecular mechanisms such as nucleosome ubiquitination that are difficult to elucidate using conventional methods. This review systematically summarizes key technological advances in split inteins over the past decade, highlighting engineering strategies, mechanistic insights, and the development of orthogonal components. It comprehensively surveys emerging applications at the frontiers of protein research, analyzes current core challenges, and proposes future directions, particularly emphasizing artificial intelligence-driven de novo design and novel splicing pathways to break existing technical bottlenecks. By enabling traceless, efficient, and versatile protein manipulation, split inteins continue to serve as indispensable tools that drive innovation in protein engineering and fundamental life science research.

Tacit knowledge of tongue inspection is content that affects diagnostic results but is difficult to be explicit. Its cognitive characteristics are rooted in xiang thinking and embodiment. It constructs multidimensional associations between tongue manifestations and disease mechanisms through imagery mapping， and it relies on the practitioner's bodily perception， clinical context， and long-term practice for continuous internalization. In response to the neglect of tacit transmission and individual cognitive processes in current traditional Chinese medicine （TCM） tacit knowledge research， an empirical research framework integrating cognitive neuroscience is proposed. Specifically， event-related potential （ERP） and eye tracking （ET） technologies can be used to capture the brain electrical activity and eye movement trajectories of experts and novices in different tongue diagnosis cognitive tasks. By integrating multimodal cognitive data such as behaviorism， ERP components， and ET indicators， the implicit cognitive advantages of experts in the perception， mapping， and decision-making stages of tongue diagnosis are presented， and the possibility of achieving tacit knowledge transformation through visual cognitive transfer training is explored. Exploring empirical pathways to uncover the cognitive mechanisms of tongue diagnosis tacit knowledge is of great significance for the inheritance of traditional Chinese medicine.

Objective To investigate the regulatory role and underlying mechanism of the long non-coding RNA (LncRNA) LINC01430 in the inhibitory effect of sanguinarine on non-small cell lung cancer (NSCLC). Methods Differentially expressed LncRNAs and coexpressed target genes in A549 and H1975 cells before and after sanguinarine treatment were identified through high-throughput lncRNA sequencing. LINC01430 expression and its prognostic value were analyzed by using the TCGA database and experimental validation. An LINC01430-overexpressing A549 cell model was established, and CCK-8, TUNEL, RT-qPCR, Western blot analysis, and dual-luciferase reporter assays were performed to evaluate the influence of LINC01430 overexpression on sanguinarine-mediated cell proliferation, apoptosis and NOX4/IL-6 expression. Results A total of 361 differentially expressed LncRNAs, among which LINC01430 was significantly downregulated (P<0.001), were identified after sanguinarine treatment. Pathway enrichment analysis revealed pathways associated with oxidative stress and inflammation, and the mRNA and protein levels of the target genes NOX4 and IL-6 were markedly reduced (P<0.05, P<0.01). LINC01430 exhibited high expression in NSCLC tissues and cell lines (P<0.05), with a trend toward poor prognosis (HR=1.15). The overexpression of LINC01430 significantly attenuated the inhibitory effect of sanguinarine on cell proliferation and the promoting effect on apoptosis in NSCLC cells (P<0.01) and partially reversed the downregulation of NOX4 and IL-6 induced by sanguinarine (P<0.05). LINC01430 directly activated IL-6 promoter transcriptional activity (P<0.05). Meanwhile, its effect on the NOX4 promoter showed an upward trend without statistical significance. Conclusion Sanguinarine may suppress NSCLC by downregulating LINC01430 to inhibit NOX4/IL-6 signaling. LINC01430 represents a promising target for NSCLC therapy.

Based on the theory of "spleen functions as wei （卫） qi"， this paper discusses the mechanism of "inflammation-metabolism-immunity" regulation in colorectal cancer cachexia and the therapeutic strategy. It is proposed that spleen deficiency with failure in transportation， leading to the maldistribution of wei （卫） qi and pathogenic constraint transforming into fire， is the source of inflammation. Spleen deficiency with failure in transportation， leading to the maldistribution of subtle essences and loss of nourishment to the physical body， is the root of metabolic depletion. Spleen deficiency with failure in transportation， leading to wei qi depletion and healthy qi decline， is the cause of immune impairment. Accordingly， a spleen-centered strategy of fortifying the spleen and restoring its defensive function is established. The specific therapeutic approaches include fortifying the spleen and boosting qi， restoring defensive function and securing the exterior to replenish the source of wei qi， with modified Sijunzi Decoction （四君子汤）， fortifying the spleen and removing toxin， resolving phlegm and stasis to restrain exuberant internal toxin and constrained fire， with modified Baitouweng Decoction （白头翁汤）， activating the spleen and harmonizing the stomach， promoting the transportation and distribution of food essence to correct nutritional and metabolic disorders， with modified Xiangsha Liujunzi Decoction （香砂六君子汤）， and fortifying the spleen and nourishing the kidney， replenishing primordial qi and consolidating the root to restore immune competence， with Buzhong Yiqi Decoction （补中益气汤） and Zuogui Beverage （左归饮） as the basic prescription.

Based on the theory of "spleen functions as wei （卫） qi"， this paper discusses the mechanism of "inflammation-metabolism-immunity" regulation in colorectal cancer cachexia and the therapeutic strategy. It is proposed that spleen deficiency with failure in transportation， leading to the maldistribution of wei （卫） qi and pathogenic constraint transforming into fire， is the source of inflammation. Spleen deficiency with failure in transportation， leading to the maldistribution of subtle essences and loss of nourishment to the physical body， is the root of metabolic depletion. Spleen deficiency with failure in transportation， leading to wei qi depletion and healthy qi decline， is the cause of immune impairment. Accordingly， a spleen-centered strategy of fortifying the spleen and restoring its defensive function is established. The specific therapeutic approaches include fortifying the spleen and boosting qi， restoring defensive function and securing the exterior to replenish the source of wei qi， with modified Sijunzi Decoction （四君子汤）， fortifying the spleen and removing toxin， resolving phlegm and stasis to restrain exuberant internal toxin and constrained fire， with modified Baitouweng Decoction （白头翁汤）， activating the spleen and harmonizing the stomach， promoting the transportation and distribution of food essence to correct nutritional and metabolic disorders， with modified Xiangsha Liujunzi Decoction （香砂六君子汤）， and fortifying the spleen and nourishing the kidney， replenishing primordial qi and consolidating the root to restore immune competence， with Buzhong Yiqi Decoction （补中益气汤） and Zuogui Beverage （左归饮） as the basic prescription.

GPR126, also known as ADGRG6, is one of the most deeply studied aGPCRs. Initially, GPR126 was thought to be a receptor associated with muscle development and was primarily expressed in the muscular and skeletal systems. With the deepening of research, it was found that GPR126 is expressed in multiple mammalian tissues and organs, and is involved in many biological processes such as embryonic development, nervous system development, and extracellular matrix interactions. Compared with other aGPCRs proteins, GPR126 has a longer N-terminal domain, which can bind to ligands one-to-one and one-to-many. Its N-terminus contains five domains, a CUB (complement C1r/C1s, Uegf, Bmp1) domain, a PTX (Pentraxin) domain, a SEA (Sperm protein, Enterokinase, and Agrin) domain, a hormone binding (HormR) domain, and a conserved GAIN domain. The GAIN domain has a self-shearing function, which is essential for the maturation, stability, transport and function of aGPCRs. Different SEA domains constitute different GPR126 isomers, which can regulate the activation and closure of downstream signaling pathways through conformational changes. GPR126 has a typical aGPCRs seven-transmembrane helical structure, which can be coupled to Gs and Gi, causing cAMP to up- or down-regulation, mediating transmembrane signaling and participating in the regulation of cell proliferation, differentiation and migration. GPR126 is activated in a tethered-stalk peptide agonism or orthosteric agonism, which is mainly manifested by self-proteolysis or conformational changes in the GAIN domain, which mediates the rapid activation or closure of downstream pathways by tethered agonists. In addition to the tethered short stem peptide activation mode, GPR126 also has another allosteric agonism or tunable agonism mode, which is specifically expressed as the GAIN domain does not have self-shearing function in the physiological state, NTF and CTF always maintain the binding state, and the NTF binds to the ligand to cause conformational changes of the receptor, which somehow transmits signals to the GAIN domain in a spatial structure. The GAIN domain can cause the 7TM domain to produce an activated or inhibited signal for signal transduction, For example, type IV collagen interacts with the CUB and PTX domains of GPR126 to activate GPR126 downstream signal transduction. GPR126 has homology of 51.6%-86.9% among different species, with 10 conserved regions between different species, which can be traced back to the oldest metazoans as well as unicellular animals.In terms of diseases, GPR126 dysfunction involves the pathological process of bone, myelin, embryo and other related diseases, and is also closely related to the occurrence and development of malignant tumors such as breast cancer and colon cancer. However, the biological function of GPR126 in various diseases and its potential as a therapeutic target still needs further research. This paper focuses on the structure, interspecies differences and conservatism, signal transduction and biological functions of GPR126, which provides ideas and references for future research on GPR126.

Objective : To characterize MMP12 as a pan-cancer marker and assess its screening value as a tumor serum marker. Methods : Bioinformatics tools such as GEPIA2,GSCA,cBioPortal,and GeneMANIA were used to analyze the pan-cancer features of MMP12 in TCGA datasets,including encompassing differential gene expression analysis,prognostic analysis,DNA methylation analysis,gene structural variation analysis and immune microenvironment analysis.Furthermore,serum samples we collected from patients with lung adenocarcinoma,breast invasive carcinoma,esophageal squamous cell carcinoma,stomach adenocarcinoma,liver hepatocellular carcinoma,and healthy individuals.ELISA was used to detect MMP12 expression in serum,and the screening performance was evaluated using the area under the ROC curve.Additionally,we followed up 28 ESCC patients and compared serum MMP12 levels between 19 patients with disease progression and 9 patients with stable disease. Results: The pancancer feature analysis revealed a significant negative correlation between MMP12 mRNA expression and its promoter DNA methylation(P<0.05),as well as a positive correlation with gene copy number variations(P<0.05).MMP12 mRNA expression was up-regulated in 14 cancer tissues compared to normal tissues next to cancer(P<0.05) and was associated with poor prognosis of cancer patients(P<0.05).Immunocorrelation analysis showed that MMP12 was significantly associated with immunity,infiltration of stromal cells,tumor mutational burden(TMB) and microsatellite instability(MSI)(P<0.05).ROC curve analysis indicated that MMP12 could serve as a potential biomarker for screening lung adenocarcinoma,breast invasive carcinoma,esophageal squamous cell carcinoma,stomach adenocarcinoma,and liver hepatocellular carcinoma.In a 30-month follow-up study of esophageal squamous cell carcinoma patients,the expression of MMP12 was higher in the disease progression group than that in the stable group. Conclusion MMP12 serves as a potential prognostic and screening marker of pan-cancer.

Objective To understand the clinical distribution characteristics and drug-resistance genotypes of carbapenem-resistant Pseudomonas aeruginosa(CRPA)in a tertiary hospital in Shanghai,so as to guide the rational use of antibiotics,reduce bacterial resistance and control nosocomial infections.Methods A total of 94 consecutive and non-redundant CRPA strains isolated from clinical specimens were collected in The First Affiliated Hospital of Naval Medical University between Jan.1,and Dec.31,2019.The distribution of CRPA across departments and sample sources were analyzed.Antimicrobial susceptibility testing against 13 antibiotics was determined using the VITEK 2 Compact automated microbiology analyzer and the Kirby-Bauer disk diffusion method.Polymerase chain reaction(PCR)was employed to detect genes encoding extended-spectrum β-lactamase resistance gene,carbapenemase resistance gene,and porin resistance gene.Homology analysis of selected isolates was performed using kSNP3,a k-mer-based software,for single nucleotide polymorphism(SNP)analysis.An evolutionary tree was constructed to assess genetic relationships.Additionally,multilocus sequence typing(MLST)was performed using the Pseudomonas aeruginosa typing scheme from the PubMLST database.Results Among the 94 CRPA isolates,high resistance rates were observed for carbapenems,including imipenem(92.6%,87 strains)and meropenem(87.2%,82 strains).In contrast,low resistance rates were detected for aminoglycosides,such as amikacin(10.6%,10 strains),gentamicin(20.2%,19 strains),and tobramycin(20.2%,19 strains).The top 3 departments in terms of isolate distribution were the Emergency Intensive Care Unit(9.6%,9 strains),Department of Cerebrovascular Surgery(8.5%,8 strains),and Department of Respiratory Medicine(8.5%,8 strains).PCR analysis of 94 CRPA strains detected outer membrane protein D2(OprD2)gene deletion in 47(50.0%)strains,13(13.8%)strains were positive for blaKPC,4(4.3%)strains for blaVIM,2(2.1%)strains for blaIMP,1(1.1%)strain for blaNDM,12(12.8%)strains for blaTEM,4(4.3%)strains for blaPER,and 2(2.1%)strains for blaGES,while blaOXA-48,blaBIC,blaSIM,blaVEB,and blaSHV were not detected.MLST identified 36 different sequence types(STs),with ST463 and ST274 being the most common,and 2 new ST(ST4023 and ST4024)were identified for the first time.Conclusion CRPA strains carry multiple resistance genes and exhibit concurrent resistance to several commonly used clinical antibiotics.The resistance is primarily associated with the presence of blaKPC,blaVIM and blaTEM genes and the deletion of OprD2 gene.Clinical monitoring of CRPA should be strengthened,and rational use of antimicrobial agents is essential to control its spread within the hospital.

Objective To investigate the role of poly(rC)-binding protein 1(PCBP1)in cadmium(Cd)-induced ferroptosis in mouse neuroblastoma Neuro-2a(N2A)cells.Methods N2A cells were exposed to a concentration gradient of CdCl?(0,1,2,4 μmol/L)for 72 h.Cell viability was assessed by trypan blue staining.Western blotting was employed to detect the expression of ferroptosis-related proteins(GPX4,HMOX1,ACSL4)and PCBP1.Intracellular Fe2? level and lipid peroxidation were detected using FerroOrange and BODIPY581/591 C11 probes,respectively.Ferrostatin-1(Fer-1),a ferroptosis inhibitor,was applied to confirm the critical role of ferroptosis in Cd-induced cytotoxicity.Molecular docking was performed to elucidate the interaction between PCBP1 and ferritin,as well as the binding sites of Cd2?.PCBP1 overexpression plasmid was further constructed for functional validation.Results Cd exposure suppressed cell viability in N2A cells in a dose-dependent manner(P<0.01),significantly down-regulated GPX4 expression(P<0.05),up-regulated HMOX1 expression(P<0.01),and induced Fe2? overload and lipid peroxidation(P<0.01).Molecular docking revealed that Cd2? directly bound to the KH2 domain of PCBP1 and then co-localized on the outer surface of ferritin heavy chain.Overexpression of PCBP1 markedly reversed Cd-induced Fe2? accumulation,GPX4 down-regulation,lipid peroxidation,and cell death.Conclusion Cd exposure disrupts PCBP1-mediated iron homeostasis via transcriptional suppression and competitive displacement of metal ions,and then synergistically drives Fe2? overload-triggered ferroptosis cascades,ultimately leading to neurotoxicity.Targeting PCBP1-mediated iron homeostasis can effectively mitigate Cd-induced neurotoxicity,and may serve as a novel therapeutic strategy.

Liquiritigenin(LG)is a flavone of pharmacological importance,however,its application potential is severely limited due to its poor water solubility.LG could be disassociated slightly in water to form phenolate anion,therefore,better solubilization effect is expected by inclusion with cationic cyclodextrins(CCDs).In this work,four kinds of CCDs modified with amino groups at the primary face were synthesized,and their solid inclusion complexes with LG were successfully prepared by preparing their saturated solutions.The formation of the solid inclusion complexes was confirmed by scanning electron microscopy(SEM)and powder X-ray diffraction(PXRD),and their supramolecular binding behavior in solution was studied using multiple techniques.A 1∶1 inclusion stoichiometry of inclusion complexation was defined using Job plot by ultraviolet-visible(UV-vis)spectroscopy,and their binding stability constants(Ks)were determined as 2862.77,3494.70,6521.85 and 9599.48 L/mol using UV-vis spectroscopic titration,far more superior to that of nativeβ-CD(Ks=236.79 L/mol).This indicated that the amino side chains on CCDs could actively participate in the inclusion complexation through anion-cation interactions,significantly strengthening the host-guest binding between CCDs and LG.The inclusion modes were further elucidated based on proton and two-dimensional rotating-frame overhauser enhancement spectroscopy(2D-ROESY)nuclear magnetic resonance(NMR)experiments and molecular docking.Water solubility of LG was dramatically promoted up to 4.9 mg/mL,which was 70-fold higher than that of native LG.This study could draw inspiration for the binding and solubilization of phenols such as flavones by design of cationic macrocyclic molecules.